Method of detection and disease state management for renal diseases

ABSTRACT

The present invention relates to a method of detection of an early-stage renal disease, comprising determining the concentration of human lipocalin-type prostaglandin D synthase in a body fluid sample taken from a subject and comparing the determined concentration with a reference value set by determining the concentrations of human lipocalin-type prostaglandin D synthase in body fluid samples taken from healthy subjects; and a method of disease state management for a renal disease, comprising determining the concentration of human lipocalin-type prostaglandin D synthase in a body fluid sample taken from a subject and evaluating the glomerular filtration ability of the subject from the determined concentration.

TECHNICAL FIELD

The present invention relates to a method for detecting renal diseases.More specifically, the invention relates to a method for detectingearly-stage renal diseases which are undetectable by existing diagnosismethods; and a method for managing the disease state of renal diseasesby evaluating the glomerular filtration ability of patients in a simplemanner.

BACKGROUND ART

Recently, the number of patients who begin to undergo dialysis therapybecause of diabetic nephropathy is increasing year by year. According tostatistical data in 1994, the number of patients with diabeticnephropathy who have newly started dialysis therapy in one year was9,351, which is very close to the corresponding number 10,995 inpatients with chronic glomerulonephritis (Akira Sekikawa et al., The41^(st) Annual Meeting of Japan Diabetes Society) Unfortunately,however, dialysis therapy for diabetic patients causes many problemssuch as cardiac failure, raising or lowering of blood pressure,infections or shunt troubles, and the average duration of life after theintroduction of dialysis is as short as about 3 years (see, for example,Statistical Survey Committee of the Japanese Society for DialysisTherapy, Journal of the Japanese Society for Dialysis Therapy, 25,1095–1103, 1992). Thus, it is believed that early treatment by earlydiagnosis is important for diabetic nephropathy.

In diagnosis and disease state management for renal diseases such asdiabetic nephropathy and glomerulonephritis that cause glomerularlesions, clearance tests using inulin, creatinine, etc. are consideredbo be useful for the evaluation of glomerular filtration ability.However, clearance tests require timed urine. Also, in clearance testswhere an exogenous substance such as inulin is administered, intravenousinjection of the exogenous substance is required. Therefore, due to thetime and labor required for such tests, application of a clearance testhas been limited to patients with apparent renal diseases. Accordingly,it is rather rare to use a clearance test as a routine diagnosis methodfor those patients who are suspected of renal diseases or who maypossibly have renal diseases. In many cases, diseases of such patientshave been diagnosed by detecting persistent proteinuria or determiningserum creatinine concentrations. However, it is also known thatirreversible glomerular lesions have already progressed when persistentproteinuria appears or when serum creatinine concentrations increase(see, for example, “Nephrology”, Kiyoshi Kurokawa (ed.), 249–251, 1995).Therefore, the detection of persistent proteinuria or the determinationof serum creatinine concentration cannot be effective means to detectearly-stage lesions.

Recently, it has become possible to determine various proteins excretedinto urine. For example, it has become clear that albumin excretion inurine increases in diabetic nephropathy, i.e. microalbuminuria precedespersistent proteinuria. At present, it is a general practice to diagnoserenal diseases with the appearance of microalbuminuria or the like.

Generally, diabetic nephropathy and other nephropathy are classifiedinto the 1st stage (pre-nephropathy stage), the 2nd stage (earlynephropathy stage), the 3rd stage (apparent nephropathy stage), the 4thstage (renal failure stage) and the 5th stage (dialysis therapy stage)(Yukio Shigeta et al., “1991 Diabetes Survey Report”, Ministry of Healthand Welfare, 317–320, 1992). The stage at which microalbuminuria isdetected falls under the above-described 2nd stage (early nephropathystage). Pathologically, mild to medium grade diffuse lesions are alreadypresent at this stage; the presence of nodular lesions is also known atthis stage. For example, diabetic nephropathy is characterized bythickening of the glomerular basement membrane and expansion of themesangial region. However, it is known that these changes are alreadypresent even at a stage when no microalbuminuria appeares clinically(Shigeki Inomata et al., Journal of the Japan Diabetes Society, 30,429–435, 1987; Bangstad, H., J. et al., Diabetologia, 36, 523–529,1993). Therefore, the 2nd stage (early nephropathy stage) does notnecessarily mean the beginning of nephropathy, and is understood as thestage at which nephropathy becomes diagnosable by current clinicaltests. On the other hand, it is believed that those abnormalitiesoccurring at the 1st stage (pre-nephropathy stage) prior to theappearance of microalbuminuria can only be detected by renal biopsy.However, since renal biopsy is invasive, it involves pain and danger.Besides, it requires enormous labor and time from the beginning ofbiopsy to the obtainment of results. Therefore, the development of asimple, non-invasive test method is desired which can detect thoseabnormalities occurring prior to the appearance of microalbuminuria.

Under such circumstances, it was reported that urinary type-IV collagenexcretion increases even at the pre-nephropathy stage, and thepossibility that urinary type-IV collagen could be an earlier indicatorof diabetic nephropathy has been suggested (Hayashi, Y. et al., DiabeticMedicine, 9, 366–370, 1992; Yagame, M. et al., J. Clin. Lab. Anal., 11,110–116, 1997). It is believed that such increase of urinary type-IVcollagen excretion reflects histological changes such as thickening ofthe glomerular basement membrane and mesangial expansion, and that theincrease is the result of enhanced production of type-IV collagen in theglomerular basement membrane, glomerular epithelial cells or tubularepithelial cells (Motohide Isono et al., Journal of the Japan DiabetesSociety, 39, 599–604, 1996). However, it is considered that even beforethe above-mentioned histological changes take place in the glomerulus,various changes in metabolism, morphology, etc. are occurring at thecell level, corresponding to hyperglycemia characteristic in diabetes.For example, up-regulation of protein kinase C activity has beenreported in the glomeruli of diabetic rats, and glomerular or mesangialcells cultured under high glucose concentrations. The relation betweenthis rise and histological changes in diabetic complications such asdiabetic nephropathy is attracting attention (Craven, P., A. andDeRubertis, F. R., J. Clin. Invest., 83, 1667–1675, 1989; Williams, B.and Schrier, R. W., Diabetes, 41, 1464–1472, 1992). Therefore, if it ispossible to detect these qualitative changes at the cellular levelearlier, more effective treatment could be given before histologicalchanges have occurred. However, no reports have been made to date whichclinically examine such utility.

On the other hand, human lipocalin-type prostaglandin D synthase(hereinafter, referred to as “L-PGDS”) is an enzyme which catalyzes theisomerization of PGH₂ (a common precursor of various prostaglandins) toPGD₂ that exhibits various physiological actions such as sleep induction(Urade, Y., Fujimoto, N. and Hayaishi, O., J. Biol. Chem., 260,12410–12415, 1985; Urade, Y., Watanabe, K. and Hayaishi, O., J. LipidMediator Cell Signaling, 12, 257–273, 1995). Recently, it was revealedthat this L-PGDS is identical with β-trace which was known to be presentin human cerebrospinal fluid (CSF) abundantly (Hoffmann, A., Conradt, H.S., Gross, G., Nimitz, M., Lottspeich, F., and Wurster, U., J.Neurochem., 61, 451–456, 1993; Zahn, M. Mader, M., Schmidt, B.,Bollensen, E. and Felgenhauer, K., Neurosci. Let., 154, 93–95, 1993;Watanabe, K., Urade, Y., Mader, M., Murphy, C. and Hayaishi, O.,Biochem. Biophys. Res. Commun., 203, 1110–1116, 1994).

In 1969 when L-PGDS was still called β-trace, Ericsson et al. publisheda paper suggesting correlation between renal diseases and β-trace(L-PGDS) (Ericsson, J., Link, H. and Zettervall, O., Neurology, 19,606–610, 1969). Since assay methods at that time were technicallyimmature, L-PGDS was undetectable in serum and urine from healthysubjects, but it was detected in serum and urine from patients withrenal diseases such as chronic glomerulonephritis in which abnormalitiesare recognized in serum creatinine concentration and creatinineclearance. It was suggested that L-PGDS concentrations increase in thosepatients. Felgenhauer et al. also reported that serum L-PGDS, which wasnot detected in healthy subjects, was detected though in only onepatient with renal failure (Felgenhauer, K., Schadlich, H. J. and Nekic,M., Klin. Wochenschr., 65, 764–768, 1987). Against these findings,Whitsed et al. developed a more sensitive assay system, compared urinaryL-PGDS concentrations (amounts excreted/24 hr) from healthy subjects andthose from patients with renal diseases presenting proteinuria, andreported that patients with renal diseases not necessarily exhibitedhigher L-PGDS concentrations (Whitsed, H., and Penny, R., Clin. Chim.Acta, 50, 111–118, 1974). It is believed that the reason why researchershave such opposite opinions on the correlation between renal diseasesand L-PGDS is because the assay system used in these reports aresemi-quantitative methods based on classical immunological methods usingpolyclonal antibodies. Recently, using more quantitative assay system,Hoffmann et al. have confirmed that serum L-PGDS concentrations inpatients with end-stage renal failure (at dialysis therapy stage) areremarkably increased as compared to the concentrations in healthysubjects (Hoffmann, A., Nimtz, M. and Conradt, H. S., Glycobiology, 7,499–506, 1997). However, although they use monoclonal antibodies withclarified specificity, their assay method is complicated. Briefly, theypurify L-PGDS from serum samples, and then compare the strength of thebands on Western blot. Thus, it is considered to be still difficult toaccurately compare minor differences in concentration by this method.

As described above, any of the examinations concerning the correlationbetween renal diseases and L-PGDS made to date has merely detected aremarkable rise of L-PGDS concentrations in those patients who have beenconfirmed to have an evidently advanced renal disease by existingdiagnostic methods, e.g. showing abnormality in creatinine kinetics,presenting proteinurea or being at dialysis therapy stage. On thecontrary, no examinations have been made to date concerning L-PGDSconcentrations prior to the progress of renal diseases.

DISCLOSURE OF THE INVENTION

It is an object of the invention to provide a method which can detectthose renal abnormalities occurring prior to early-stage nephropathythat were undetectable by various diagnostic methods so far employed,accurately and without imposing a big burden on subjects. Further, it isanother object of the invention to provide a method which can evaluatesimply and in a short time glomerular filtration ability that has beenevaluated so far by clearance tests requiring much time and labor.

The inventors have made intensive and extensive researches toward thesolution of the above-mentioned problems. As a result, the inventorshave found that it is possible to detect renal diseases prior to theearly nephropathy stage at which diseases have progressed to some extentby determining L-PGDS concentrations in body fluid samples (e.g., serum,urine) and using the determined values as an indicator, and that it isalso possible to manage the disease state of renal disease patients byusing the determined values as an indicator. Thus, the present inventionhas been achieved.

The present invention relates to a method of early detection of a renaldisease, comprising determining the concentration of L-PGDS in a bodyfluid sample taken from a subject and comparing the determinedconcentration with a reference value set by determining theconcentrations of L-PGDS in body fluid samples taken from healthysubjects.

The present invention also related to a method of disease statemanagement for a renal disease, comprising determining the concentrationof L-PGDS in a body fluid sample taken from a subject and evaluating theglomerular filtration ability of the subject from the determinedconcentration.

Hereinbelow, the present invention will be described in detail.

In the present invention, samples to be used for L-PGDS assay are bodyfluid samples taken from subjects. Specifically, blood (serum orplasma), urine (spotted urine, timed urine, etc.), amniotic fluid, orthe like may be used.

As a method for determining L-PGDS concentrations in the above-mentionedsamples, any assay method may be used as long as it can determine L-PGDSconcentrations accurately. For example, an immunological assay method oran enzyme activity assay method may be used. However, from the viewpointof necessity to handle a large number of samples at one time and in asimple manner at actual clinical sites, it is preferable to employ animmunological assay method such as EIA, ELISA, RIA or FIA using L-PGDSspecific monoclonal antibodies or polyclonal antibodies.

Of the above-described immunological assay methods, sandwich ELISA usingL-PGDS specific monoclonal antibodies is especially preferable. Specificexamples of the monoclonal antibodies include those antibodies producedby hybridoma cell strains 1B7 (FERM BP-5709), 7F5 (FERM BP-5711), 6F5(FERM BP-5710), 9A6 (FERM BP-5712) and 10A3 (FERM BP-5713).

In the determination of L-PGDS concetration by sandwich ELISA, a L-PGDSdetection kit established by the present inventors containing theabove-described monoclonal antibodies may be used (WO 97/16461).

In the present invention, it is possible to detect an early-stage renaldisease in a subject by using as an indicator the L-PGDS concentrationdetermined by the above-mentioned means. Also, it is possible to managethe disease state of a subject's renal disease by evaluating theglomerular filtration ability of the subject using as an indicator theL-PGDS concentration determined by the above-mentioned means.

In the method of the invention, a renal disease is detected by comparingthe L-PGDS concentration in a body fluid sample of a subject determinedby the above-mentioned means with a reference value set by determiningL-PGDS concentrations in body fluid samples of healthy subjects by thesame means.

The term “reference value” used herein means the value to judge positiveor negative. This value is also called the “cut-off value” at clinicalsite. This reference value may be set, for example, by the followingformula:

Mean value in healthy subjects+(α×standard deviation) wherein α may be0.5, 1, 2, 5 or the like. 2 is often used for α In populations whichshow regular distribution.

Specifically, the detection of a renal disease is performed as follows.First, L-PGDS concentrations in body fluid samples are determined on apopulation composed of healthy subjects to set a reference value by theabove-described formula. Subsequently, if the L-PGDS concentration in asubject body fluid sample is above the reference value (i.e. showsabnormal value), the subject is judged positive.

Disease state management for a renal disease is performed by evaluatingthe glomerular filtration ability of a subject using the L-PGDSconcentration in a body fluid sample of the subject determined by theabove-mentioned means.

In the present invention, the term “disease state management” means tograsp the condition of a disease (the degree of severity) and to predictthe behavior of the disease (prognosis).

As renal diseases which can be detected by the method of the inventionor renal diseases of which the disease state can be managed by themethod of the invention, renal diseases accompanied with glomerularlesions, renal diseases associated with hypertension, renal diseasesassociated with lipid metabolic disorder or the like may be enumerated.Specific examples include nephropathy, etc. More specifically,glomerulonephritis, nephrotic syndrome, diabetic nephropathy, polycystickidney, renal failure or the like may be enumerated. The term “diabeticnephropathy” used herein include diabetic nephropathy of extremely earlystage, for example, before microalbuminuria appears or before urinarytype-IV collagen increases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows correlation between serum L-PGDS concentration and serumcreatinine concentration.

FIG. 2 shows correlation between urinary L-PGDS index and urinaryalbumin index.

FIG. 3 shows correlation between urinary L-PGDS index and urinarytype-IV collagen index.

FIG. 4 shows serum L-PGDS concentration and serum creatinineconcentration (Panel A) and urinary L-PGDS index and urinary albuminindex (Panel B) in patients with various renal diseases.

FIG. 5 shows correlation between serum L-PGDS concentration/serumcreatinine concentration and the history of diabetes (Panel A) andcorrelation between urinary L-PGDS index/urinary albumin index and thehistory of diabetes (Panel B).

FIG. 6 shows correlation between serum L-PGDS concentration andcreatinine clearance (Panel A) and correlation between urinary L-PGDSindex and creatinine clearance (Panel B).

FIG. 7 shows changes in urinary L-PGDS index and urinary type-IVcollagen index at various albuminuria stages classified based on urinaryalbumin index.

FIG. 8 shows positive ratios judged by urinary L-PGDS index and urinarytype-IV collagen index at various albuminuria stages classified based onurinary albumin index.

FIG. 9 shows positive ratios judged by urinary L-PGDS index in a groupof diabetic patients positive in type-IV collagen index and a group ofdiabetic patients negative in this index.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in more detail withreference to the following Examples, which should not be construed aslimiting the scope of the invention.

REFERENCE EXAMPLE 1 Determination of Serum and Urinary L-PGDSConcentrations in Healthy Persons and the Setting of Reference Values

Using blood (serum) samples and spotted urine samples taken from healthysubjects, L-PGDS concentrations in these body fluid samples weredetermined by sandwich ELISA to set reference values.

(1) Preparation of A Standard Curve

First, an anti-L-PGDS monoclonal antibody (clone: 7F5) which can bind toL-PGDS was diluted with 50 mM carbonate buffer (pH 9.6) to give aconcentration of 4.4 μg/ml. Then, this diluted antibody was added to a96-well microtiter plate at 300 μl/well and left overnight at 4° C. tothereby immobilize the antibody on the plate. This plate was washed withphosphate buffered saline (pH 7.4; hereinafter called “PBS”) threetimes. Thereafter, 0.2% casein-containing PBS (pH 7.4; hereinaftercalled the “blocking solution”) was added to the plate at 300 μl/welland incubated at 30° C. for 90 min for blocking.

Subsequently, the plate after blocking was washed with 0.05% Tween20-containing PBS (T-PBS) three times. Then, 100 μl of reference L-PGDSsolution (which was prepared by stepwise dilution of L-PGDS purifiedfrom CSF with the blocking solution) was added to each well andincubated at 30° C. for 90 min. After the reaction, the plate was washedwith T-PBS three times. Then, 100 μl of horseradish peroxidase-labelledanti-L-PGDS monoclonal antibody (clone: 1B7) diluted with the blockingsolution to give a concentration of 0.5 μg/ml was incubated at 30° C.for 90 min. After the plate was washed with T-PBS three times, 100 μl ofa chromogenic substrate solution (ABTS solution; Boehringer Mannheim)was added to each well and incubated at 30° C. for 30 min. Then, 100 μlof a stopping solution (1.5% oxalic acid) was added to each well andagitated with a plate mixer to terminate the reaction. Using acommercial plate reader (model SK601; manufactured by Seikagaku Corp.),the difference between the absorbances at 405 nm and 490 nm (A405nm–A490 nm) was determined to thereby prepare a reference curve.

The monoclonal antibodies (clones: 1B7 and 7F5) used in the abovesandwich ELISA were obtained as follows. Briefly, 1.0 ml of pristan wasinjected into mice intraperitoneally. Two weeks later, eachantibody-producing hybridoma cell strain was transplanted into theperitoneal cavity of the mice (1×10⁸ cells/mouse). Two weeks thereafter,ascites fluid was collected from each mouse and subjected to protein Aaffinity column chromatography to obtain the monoclonal antibody ofinterest (3–10 mg/ml).

The designations of the above-mentioned monoclonal antibody-producinghybridoma cell strains coincide with the designations of theabove-mentioned monoclonal antibodies. These cell strains, 1B7 and 7F5,were deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1–3,Higashi 1-chome, Tsukuba City Ibaraki Pref., Japan) under the AccessionNumbers of FERM BP-5709 (original deposit date: Sep. 21, 1995) and FERMBP-5711 (original deposit date: Jun. 6, 1996), respectively.

(2) Determination of L-PGDS Concentrations in Samples

One hundred and ninety-two serum samples and 56 spotted urine sampleswere used. After these samples were appropriately diluted with theblocking solution, L-PGDS concentrations were determined by the sandwichELISA as described above. The results of the assay revealed that themean value±standard deviation of serum L-PGDS concentration obtainedfrom healthy subjects was 0.848±0.186 μg/ml. In the analysis usingspotted urine samples, the determined values were converted into urinaryL-PGDS indexes (L-PGDS/g creatinine) using urinary creatinineconcentrations, considering influences of the difference in urinaryconcentrations depending on the time of sampling. As a result, the meanvalue±standard deviation of urinary L-PGDS index obtained from healthysubjects was 2.44±1.86 mg/g creatinine. From the thus obtained meanvalue±standard deviation, a reference value was set according to theformula described earlier, i.e. the mean value+(2×standard deviation).The reference value for serum L-PGDS concentration was 1.22 μg/ml andthe reference value for urinary L-PGDS index was 6.16 mg/g creatinine.

In the following Examples, L-PGDS in body fluid samples (i.e. serum andurine) was determined in the same manner as described in the ReferenceExample; creatinine in those samples was determined by the alkalinepicrate method; urinary albumin was determined by the latexagglutination method; and urinary type-IV collagen was measured by thesandwich ELISA. Since spotted urine was used as urine samples,determined values from these samples were converted into values per gramof creatinine in urine taking the difference in urinary concentrationsinto consideration (urinary L-PGDS index: L-PGDS/g creatinine; urinaryalbumin index: albumin/g creatinine; urinary type-IV collagen index:type-IV collagen/g creatinine).

EXAMPLE 1 Correlation Between L-PGDS and Serum Creatinine/UrinaryAlbumin/Urinary Collagen IV

Serum L-PGDS concentration, serum creatinine concentration, urinaryL-PGDS index and urinary albumin index were determined on 118 internalmedicine outpatients. Also, urinary L-PGDS index and urinary type-IVcollagen index were determined on 284 internal medicine outpatients.

As shown discretely in FIGS. 1 to 3, a high correlation was foundbetween serum L-PGDS concentration and serum creatinine concentration,between urinary L-PGDS index and urinary albumin index and betweenurinary L-PGDS index and urinary type-IV collagen index. Therefore, ithas become evident that L-PGDS can be an indicator of renal diseaseswhich is comparable to serum creatinine, urinary albumin and urinarytype-IV collagen used so far.

EXAMPLE 2 Changes in L-PGDS Concentrations in Patients with VariousRenal Diseases

Serum L-PGDS concentration and urinary L-PGDS index were determined on42 patients with renal diseases (chronic renal failure: 11 patients;glomerulonephritis: 23 patients; polycystic kidney: 8 patients). At thesame time, serum creatinine concentration and urinary albumin index weredetermined. The results are shown in FIG. 4.

As shown in FIG. 4, it has become clear that in all of the renaldiseases tested, serum L-PGDS concentration and urinary L-PGDS indexincrease significantly compared to those values in healthy subjects.Serum creatinine concentration and urinary albumin index also increasedin those renal diseases. However, comparing the p values (levels ofsignificance), L-PGDS showed smaller p values. Therefore, we can saythat L-PGDS can be an indicator of renal diseases superior to serumcreatinine and urinary albumin.

EXAMPLE 3 Changes in L-PGDS Concentrations in Diabetic Patients

Serum L-PGDS concentration and uninary L-PGDS index were determined on55 diabetic patients. At the same time, serum creatinine concentrationand urinary albumin index were determined. The patients were classifiedaccording to the history of their diabetes (less than 5 years: 22; from5 years to less than 10 years: 19; 10 years or more: 14). The resultsare shown in FIG. 5.

As shown in FIG. 5, it was found that the longer the disease history is,the more both serum L-PGDS concentration and uninary L-PGDS indexincrease. Serum creatinine concentration and urinary albumin index alsoincreased as the disease history was longer. However, comparing the pvalues (levels of significance), L-PGDS showed smaller p values. Sincediabetes easily becomes complicated by nephropathy, we can say thatL-PGDS can be an indicator of progression of diabetic nephropathysuperior to serum creatinine and urinary albumin.

EXAMPLE 4 Prospective Study of Diabetic Patients

A two-year prospective study was performed on the 6 patients who werenormal in both serum creatinine concentration and urinary albumin indexbut abnormal in serum L-PGDS concentration and/or urinary L-PGDS indexin Example 3. In this study, abnormal values were defined as follows. Asto L-PGDS, the mean value +(2×standard deviation) obtained from healthysubjects (i.e., 1.22 μg/ml for serum L-PGDS concentration and 6.16 mg/gcreatinine for urinary L-PGDS index) or more was considered abnormal. Asto serum creatinine concentration, 1.1 mg/dl or more was consideredabnormal. As to urinary albumin index, 30 mg/g creatinine or more wasconsidered abnormal. The results are shown in Table 1.

TABLE 1 Follow-up Survey on Diabetic Patients Subject 1 Subject 2Subject 3 Subject 4 Subject 5 Subject 6 [At the beginning of the test]Blood creatinine concentration (mg/dl) 0.9 1.0 0.8 0.7 0.9 0.7 Urinaryalbumin index (mg/g creatinine) 15.9 28.8 15.2 19.4 8.8 10.1 BloodL-PGDS concentration (μg/ml) 1.86* 1.69* 1.25* 1.06 0.70 0.94 UrinaryL-PGDS index (mg/g creatinine) 13.66* 12.21* 5.79 14.94* 7.05* 9.80* [2years later] Blood creatinine concentration (mg/dl) 1.3* 5.5* 3.4* 0.61.1* 0.7 Urinary albumin index (mg/g creatinine) 64.9* 373.8* 269.0*29.1 103.2* 8.8 Blood L-PGDS concentration (μg/ml) 2.03* 6.91* 1.73*1.14 1.37* 1.12 Urinary L-PGDS index (mg/g creatinine) 14.60* 17.57*19.59* 5.10 7.75* 6.72* *Abnormal values

As shown in Table 1, all of the 6 patients were normal in both serumcreatinine concentration and urinary albumin index at the beginning ofthe test. Two years later, however, 4 patients out of the 6 exhibitedabnormal values in both serum creatinine concentration and urinaryalbumin index, showing clearly that they developed nephropathy. Thisindicates that extremely early-stage renal disease(s) which isundetectable by current clinical diagnosis can be detected bydetermining L-PGDS concentrations. As to determined concentrations, ithas become clear that it is appropriate to judge those subjects positivewho show the mean value+(2×standard deviation) obtained from healthysubjects or greater than this value.

EXAMPLE 5 Prospective Study of Internal Medicine Outpatients

A two-year prospective study was performed on the 8 patients who werenormal in both serum creatinine concentration and urinary albumin indexbut abnormal in serum L-PGDS concentration and/or urinary L-PGDS indexin Example 1. In this study, abnormal values were defined as follows. Asto L-PGDS, the mean value +(2×standard deviation) obtained from healthysubjects (i.e., 1.22 μg/ml for serum L-PGDS concentration and 6.16 mg/gcreatinine for urinary L-PGDS index) or more was considered abnormal. Asto serum creatinine concentration, 1.1 mg/dl or more was consideredabnormal. As to urinary albumin index, 30 mg/g creatinine or more wasconsidered abnormal. The results are shown in Table 2.

TABLE 2 Follow-up Survey on Outpatients with Internal Diseases Subject 1Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8[At the beginning of the test] Blood creatinine concentration (mg/dl)0.7 0.9 1.0 0.7 0.7 0.8 0.6 0.6 Urinary albumin index (mg/g creatinine)9.5 10.0 22.1 12.3 5.8 20.5 15.9 8.8 Blood L-PGDS concentration (μg/ml)2.03* 1.68* 1.88* 1.39* 0.89 0.79 1.20 1.03 Urinary L-PGDS index (mg/gcreatinine) 16.51* 8.87* 15.55* 5.44 6.74* 11.94* 14.58* 7.62* [2 yearslater] Blood creatinine concentration (mg/dl) 1.2* 0.8 3.2* 2.7* 1.2*1.3* 0.9 0.7 Urinary albumin index (mg/g creatinine) 47.2* 9.5 523.3*633.7* 349.0* 98.5* 14.0 7.0 Blood L-PGDS concentration (μg/ml) 1.38*1.16 2.08* 3.21* 1.61* 1.23* 1.07 0.93 Urinary L-PGDS index (mg/gcreatinine) 7.93* 9.27* 16.52* 22.74* 9.60* 17.77* 13.41* 5.72 *Abnormalvalues

As shown in Table 2, all of the 8 patients were normal in both serumcreatinine concentration and urinary albumin index at the beginning ofthe study. Two years later, however, 5 patients out of the 8 exhibitedabnormal values in both serum creatinine concentration and urinaryalbumin index, showing clearly that they developed a renal disease(s).This indicates that extremely early-stage nephropathy which isundetectable by current clinical diagnosis can be detected bydetermining L-PGDS concentrations. As to determined concentrations, ithas become clear that it is appropriate to judge those subjects positivewho show the mean value+(2×standard deviation) obtained from healthysubjects or greater than this value.

EXAMPLE 6 Disease State Management for Glomerular Lesions

Creatinine clearance (24 hr), serum L-PGDS concentration and urinaryL-PGDS index were determined on patients with glomerulonephritis andpatients with chronic renal failure (total 11 patients). For thedetermination of urinary L-PGDS index, spotted urine samples were used.The results are shown in FIG. 6.

As shown in FIG. 6, both serum L-PGDS concentration and urinary L-PGDSindex exhibited a high correlation with creatinine clearance. Thus, ithas become clear that the determination of L-PGDS concentration isuseful for the evaluation of glomerular filtration ability. Since thedetermination of L-PGDS concentration does not require timed urine noradministration of an exogenous substance to be cleared, this method hasmade it possible to manage the disease state of glomerular lesionssimply.

EXAMPLE 7 Progress of Diabetic Nephropathy and L-PGDS Concentration

Urinary L-PGDS index was determined on 101 diabetic patients. At thesame time, urinary albumin index and urinary type-IV collagen index werealso determined. The degrees of progress of diabetic nephropathy wereclassified into normoalbuminuria stage (urinary albumin index<30 mg/gcreatinine; 57 patients), microalbuminuria stage (30 mg/g creatinine≦urinary albumin index<300 mg/g creatinine; 27 patients) andoverproteinuria stage (urinary albumin index≧300 mg/g creatinine; 17patients). The results are shown in FIG. 7.

As shown in FIG. 7, it has become clear that both urinary L-PGDS indexand urinary type-IV collagen index increase as diabetic nephropathyprogresses. Therefore, we can say that urinary L-PGDS can be anindicator of early-stage diabetic renal diseases which is comparable tourinary albumin or urinary type-IV collagen.

Further, it was found that although the urinary L-PGDS indexes ofdiabetic patients at normoalbuminuria stage were significantly higherthan those of healthy subjects, such significant difference is notrecognized in urinary type-IV collagen indexes. This indicates thatextremely early-stage diabetic nephropathy which is undetectable bydetermining urinary albumin or urinary type-IV collagen can be detectedby determining urinary L-PGDS.

EXAMPLE 8 Progress of Diabetic Nephropathy and Positive Ratios

Positive ratios based on urinary L-PGDS index and urinary type-IVcollagen index were examined in those patients at various albuminuriastages described in Example 7. In this examination, abnormal values weredefined as follows. As to urinary L-PGDS index, 6.16 mg/g creatinine ormore was considered abnormal according to Example 5. As to urinarytype-IV collagen index, 3.7 μg/g creatinine or more was consideredabnormal according to the standard set by Isono et al. (Motohide Isonoet al., J. Jpn. Diab. Soc., 39, 599–604, 1996). For each index, patientsshowing an abnormal value were judged positive. The results are shown inFIG. 8.

As shown in FIG. 8, the patients at overproteinuria stage were 100%positive based on both urinary L-PGDS index and urinary type-IV collagenindex. However, in the patients at microalbuminuria stage, the positiveratio based on urinary L-PGDS index was slightly higher than thepositive ratio based on urinary type-IV collagen index (74.1% vs.70.4%). In the patients at normoalbuminuria stage, the positive ratiobased on urinary L-PGDS index was greatly higher than the positive ratiobased on urinary type-IV collagen index (57.9% vs. 36.8%).

Further, these 101 diabetic patients were classified into two groups(i.e. positive group and negative group) with the reference value forurinary type-IV collagen index, and then positive ratio based on urinaryL-PGDS index was examined in each group. The results are shown in FIG.9.

As shown in FIG. 9, in the urinary type-IV collagen index positivecases, more than 90% were also judged positive in urinary L-PGDS index.Thus, the results from both indexes were almost consistent with eachother. On the other hand, 40% of the urinary type-IV collagen indexnegative cases were judged positive in urinary L-PGDS index.

These results indicate that extremely early-stage diabetic nephropathywhich is undetectable by determining urinary albumin or urinary type-IVcollagen can be detected by determining urinary L-PGDS.

EXAMPLE 9 Retrospective Study of Diabetic Patients

L-PGDS index, albumin index and type-IV collagen index were determinedon those 63 patients from the subjects in Example 7 whose urine sampleshad been taken 2 to 3 years prior to the present test and stored at −20°C. In this study, abnormal values were defined as follows. As to urinaryalbumin index, 30 mg/g creatinine or more was considered abnormalaccording to the diagnostic standard set by the joint committee of theJapan Diabetes Society and the Japanese Society of Nephrology. As tourinary L-PGDS and type-IV collagen indexes, abnormal values weredefined in the same manner as in Example 8.

The results have revealed that there are 8 patients who are normal inboth urinary albumin index and urinary type-IV collagen index in storedurine samples but judged positive in urinary L-PGDS index alone. Forthese 8 patients, past (2 to 3 years ago) and present urinary L-PGDSindexes, albumin indexes and type-IV collagen indexes are summarized inTable 3.

TABLE 3 Retrospective Survey on Diabetic Patients Subject 1 Subject 2Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 [In thepast] Albumin index (mg/g creatinine) 9.6 10.9 2.3 18.9 6.7 0.6 8.8 5.7Type-IV collagen index (μg/g creatinine) 2.6 1.5 1.2 2.2 1.1 2.7 3.0 3.5L-PGDS index (mg/g creatinine) 6.60* 9.68* 10.34* 9.08* 6.43* 7.89*7.09* 6.79* [At present] Albumin index (mg/g creatinine) 55.3* 234.4*51.1* 12.3 39.5* 31.0* 9.9 31.8* Type-IV collagen index (μg/gcreatinine) 9.1* 22.8* 7.3* 11.6* 2.6 10.0* 3.7* 3.8* L-PGDS index (mg/gcreatinine) 9.41* 16.12* 13.44* 8.12* 6.28* 16.29* 4.93 10.21* *Abnormalvalues

As seen from past and present urinary L-PGDS index data, only onepatient (Subject 7) has achieved a normal value and the remaining sevenpatients still show an abnormal value in this index. Of these sevenpatients, one (Subject 5) has begun to show an abnormal value in urinaryalbumin index also and two (Subjects 4 and 7) have begun to show anabnormal value in urinary type-IV collagen index also in two to threeyears. In addition, five patients (Subjects 1, 2, 3, 6 and 8) have begunto show an abnormal value in both urinary albumin index and urinarytype-IV collagen index.

These results indicate that extremely early-stage diabetic nephropathywhich is undetectable by determining urinary albumin or urinary type-IVcollagen can be detected by determining urinary L-PGDS.

INDUSTRIAL APPLICABILITY

According to the present invention, there is provided a method which candetect renal abnormalities more remarkably than serum creatinine andurinary albumin which are used as an indicator of renal diseasesaccompanied with glomerular lesions, such as glomerulonephritis anddiabetic nephropathy. Furthermore, the method of the invention candetect, with extreme accuracy and less burden to subjects, even thoserenal diseases prior to early nephropathy stage which exhibit noabnormality in serum creatinine, urinary albumin and urinary type-IVcollagen and are undetectable by current clinical diagnosis.Additionally, according to the method of the invention, glomerularfiltration ability can be evaluated simply without timed urine oradministration of an exogenous substance to be cleared. Thus, the methodof the invention is extremely useful for detection of early-stage renaldiseases and disease state management for patients with such diseases.

1. A method of detection of an early-stage renal disease, the methodcomprising: determining the concentration of albumin in a urine sampleof a test subject to be normal, the first determination failing todetect early-stage renal disease in the subject; and determining aconcentration of human lipocalin-type prostaglandin D synthase (L-PGDS)then in a urine sample taken from the same test subject, wherein ahigher concentration of human L-PGDS in the urine sample taken from thetest subject, compared to a reference value of human L-PGDSconcentration in urine, is an indication that the test subject hasearly-stage renal disease, wherein the reference value is obtained bydetermining the concentration of human L-PGDS in urine of healthysubjects.
 2. The method of claim 1, wherein the determination of theconcentration of human L-PGDS in a urine sample from the test subject isperformed by an immunological assay.